D Pocket

The family of cytochrome P450 enzymes (CYPs) plays a central role in the metabolism of a tvide variety of xenobiotics including clinically important drugs. A number of approaches such as 3D-QSAR and pharmacophore modeling have been used to predict inhibitory potential and metabolism of drug candidates [40-45], 

In the score plots obtained, the first component discriminates between CYP2C8 and the other three enzymes, meaning that CYP2C8 is the most different among the CYP2C enzymes. The second component discriminates CYP2C18, and the third component CYP2C9 from the rest ofthe proteins, respectively. 

Nevertheless, the regions conferring selectivity towards CYP2C9 could be used to construct a receptor-pharmacophore model. This model agreed nicely with the calculated binding mode of diclofenac pointing its aromatic 4 position towards the heme. Hydroxylation of this position is specific for CYP2C9. 

More recently, Afzelius and co-workers used GRID/CPCA for a comparative analysis of protein structures of GYP2C9 and CYP2C5 from different sources crystal structures, homology models, and snapshots from molecular dynamics simulations [16]. The evaluation of molecular dynamics simulations by means of GRID/GPGA is an especially interesting new aspect in their publication. 

The last step incorporated snapshots from molecular dynamics simulations of CYP2C9 and CYP2C5 crystals in explicit water. They were analyzed to determine changes upon substrate binding and to investigate which parts of the cavity were more flexible and could participate in substrate recognition and access. 

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The hydroxyl group of Serinel95 acts as a nucleophile, targeting the scis-sile amide bond of the substrate. There are three principal binding pockets at the cleaving site the specificity (S) pocket, the proximal (P) pocket and the distal (D) pocket modelled from the crystal structure [12,17,18]. 

Figure 3.10. CPCA contour plot showing field differences between thrombin and trypsin for the DRY probe within the P and D pockets. Thrombin is drawn in blue, trypsin in red, the thrombin inhibitor NAPAP in gray. The yellow contours indicate regions where a hydrophobic group in a ligand would increase selectivity towards thrombin.

Figure3.12. CPCA contour plot ofthe field differences ofthrombin and factor Xa in the D pocket for the NM3 probe. The cyan contours indicate regions where interactions ofthe NM3 type would enhance selectivity towards factor Xa. Thrombin is drawn in blue, factorXa in green, and the selective factorXa inhibitor DX9065a [65] in gray.

Schweizer, E., Hoffmann-Roeder, A., Olsen, J. A., el al. (2006) Multipolar interactions in the D pocket of thrombin large differences between tricyclic imide and lactam inhibitors. Org. Biomol. Chem., 4, 2364-2375. 

Figure 3. Cartoon of the binding mode of fibrinogen 2 in thrombin with the arginine side chain in the recognition pocket, the valine side chain in the P-pocket and the leucine and phenylalanine side chains in the D-pocket.

There is a small volume deep in the D pocket that must be occupied by a hydrophobic group (over GE3 of Trp215). 

The diaryl sulfonamide inhibitors were discovered by a screening exercise aimed at finding less basic thrombin inhibitors [44]. A crystal structure of the complex of thrombin with the R=CH3 compound 9b (BM14.1248) shows the phenyl group in the D pocket, the central tolyl group in the P pocket, and the 4-aminopyridine in the SI pocket (but not interacting directly with Aspl89) [45] [Iwt],... 

We conclude from the above examples that up to 2 kcal mol of binding energy may be obtained by placing a methyl or similar small hydrophobic group correctly in the P pocket Something similar must be true of the D pocket, although examples with X-ray validation are missing. 

High-resolution X-ray structures of thrombin in the Protein Data Bank show rather variable total numbers of water molecules, presumably according to the preferences of the depositors. A generally accepted number is around one water per amino acid [50], i.e., 300 for thrombin. Of these, the only ones of direct interest here are the conserved water at the bottom of the SI pocket (Fig. 7.9) possible waters hydrogen bonding to the -NHs of Gly216 and Gly219 and whatever waters are in the SI, P, and D pockets and are normally displaced by inhibitors. 

Plate 13 Pseudofield plot showing MIF differences between thrombin (blue) and factor Xa (green) for the GRID trimethyl ammonium (NM3) probe within the D pocket. Favourable interaction energies are shown in blue. Reference inhibitor is DX 9065 A. °... 

D pocket. The DRY, C3 and NM3 probes show the highest interaction energies for factor Xa, and therefore, these interactions are expected to be the most important ones for selectivity towards this enzyme. In addition, for trypsin the O and OS probes are particularly favourable. 

In factor Xa, the D pocket is lined by aromatic residues in addition to the negatively charged Glu97, therefore, the finding that hydrophobic or cation-Tr interactions are important again underlines the strength of our x-variable weighting procedure. 

Factor Xa is an enzyme related to thrombin that is also involved in the blood coagulation cascade. In place of the spacious D pocket of thrombin that is... 

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